We made significant progress towards both of our long term goals: 1) development of a gene therapy for intravascular thrombosis, and 2) the development of a gene therapy for recurrent arterial stenosis. In previous experiments we demonstrated that overexpression of either wild type or mutant tissue plasminogen activator (t-PA) and urokinase (UK) from non-primate endothelial cells resulted in enhanced endothelial cell fibrinolytic activity. In the past year we have extended these findings to primate endothelial cells both in vitro and in an ex vivo vascular shunt system. The expression of recombinant plasminogen activators from cultured baboon endothelial cells was fully characterized: messenger RNA levels, mass and activity of the recombinant plasminogen activators, and effect on total cellular fibrinolytic activity were all quantitated. We tested the ability of endothelial cells transduced to overexpress t-PA in a baboon model of prosthetic graft thrombosis, and found that these cells significantly reduced the amount of thrombus deposited in this model. In addition, viral vectors expressing recombinant, biologically active hirudin (a specific thrombin inhibitor) have been constructed. We will be testing these vectors for their ability to antagonize both the procoagulant and proliferative actions of thrombin. We adopted an adenoviral vector-mediated gene transfer system in order to attempt to increase the efficiency of in vivo arterial gene transfer. In both the normal sheep carotid artery and the balloon-injured rat carotid artery we found very high levels of arterial gene transfer with the adenoviral vector. These levels are sufficiently high that we may begin to consider the introduction of therapeutic genes into the vessel wall.